The present invention relates to the field of spectroscopy, and more particularly to spectroscopy of samples occupying small volumes.
Capillary electrophoresis (CE) is a separation technique based on the differential migration of charged particles in an electric field. A thin capillary (20-100 xcexcm internal diameter) is filled with an electrolyte providing a medium in which analytes can migrate through. The sample is introduced at one end of the CE unit. An electric field typically of 100-600 V cmxe2x88x921 is applied across the capillary facilitating analyte species migration according to their electrophoretic mobility (u) passing a detector as they migrate (usually UV or fluorescence) at or near the end of the capillary.
This separation technique as well as others, such as packed capillary liquid chromatography, capillary electrochromatography and super critical chromatography, require spectroscopic measurements to be made on extremely small volumes of flowing liquid samples. The typical application has a sample flowing through a fused silica capillary tube where inside diameters range from 15 to 150 micrometers and the outside diameters range from 150 to 300 micrometers. Various techniques presently are used for directing light from a suitable source into and/or through such a small volume sample cell, as well as taking the light emanating from the inside of the cell and directing it toward a light detecting or analyzing instrument to effect optical analysis or detection of samples contained in the cell. Alignment of the optical system to efficiently direct the light from the source to the capillary cell, particularly to the bore and sample therein, and/or to direct the radiation emanating from the cell to a detector, presents problems.
The underlying problem is generally related to selecting components and precisely aligning them for the purpose of directing light from a light source such as a laser to, or through, to a volume of interest which has a small cross-sectional area perpendicular to the optical access. Similar problems are associated with collecting the light that emanates from a volume of interest and directing it to a photodetector or analyzer.
An implementation described in U.S. Pat. No. 5,037,199 to Hlousek utilizes an optical scheme which attempts to solve these focusing and alignment problems. In Hlousek""s disclosure, a laser beam is focused into the lumen of a separations capillary or cell using a ball lens. The ball lens and the capillary/cell are mounted together as a unit. A lens focuses light from a source onto the ball lens. The size and shape of the light may be controlled and/or selected by placement of one or more suitably shaped apertures on axis with the source and the capillary/cell. The sphere or ball lens concentrates light by acting as a very short focal length lens to convert slowly converging light from the laser beam source to a rapidly converging cone of light that will image the source into or through the volume of interest in the cell.
A ball lens used this way suffers severe aberrations, in particular, spherical aberration and coma, making the laser focus larger than desirable. Refraction of light rays at the cylindrical outside surface of the capillary causes astigmatism and further enlargement of the focal spot. In addition, light is lost due to surface reflections at the ball-lens-to-air and air-to-capillary interfaces. Consequently, efficiency of the fluorescence excitation of the sample suffers. Similarly, the ability to efficiently collect the beam is negatively impacted.
The present invention provides an optical scheme that substantially eliminates spherical aberration and coma, thereby substantially improving collection efficiency and fluorescence rib excitation.
According to the invention, the optical scheme utilizes a laser beam focused by an optical component, such as a microscope objective, through the curved surface of a hyper-hemisphere. The hyper-hemisphere focuses the beam sharply at a known point while avoiding spherical aberration and coma.
In one embodiment, the optical scheme comprises a hyper-hemisphere and a hemisphere. Both the hyper-hemisphere and the hemisphere have a substantially planar surface. The substantially planar surface of the hyper-hemisphere is optimally located at an internal aplanatic radius where a capillary or cell can be positioned. This results in an aplanatic focus at the location of the capillary lumen whereat the spherical aberration and coma are zero. An aluminized hemispherical exterior surface of the hemisphere retro-reflects the beam, thereby providing a second pass through the sample volume.
In one implementation, each of the substantially planar surfaces of the hyper-hemisphere and the hemisphere has a groove disposed therein. The hyper-hemisphere and the hemisphere are mated by placing in contact their substantially planar surfaces. Upon mating, the grooves in the hyper-hemisphere and the hemisphere form a channel in which a fused silica capillary is placed. The lumen of the fused silica capillary is thereby located at a second aplanatic point of the hyper-hemisphere, as well as at the center of the retro-reflecting hemisphere. Fluorescent light emitted by the small excited volume of interest is collected using the same optics, and directed to a detection means using a dichroic beamsplitter by means well known to those skilled in the art. The airspace between the fused silica capillary and the groove is filled with an index-matched gel or liquid, so that the hyper-hemisphere, capillary and hemisphere become a single optical element, thereby eliminating any reflections or losses at the air/silica interface, and also avoiding astigmatism due to the cylindrical capillary boundary.
Alternatively, no groove is formed in the substantially planar surfaces. Instead, each of the substantially planar surfaces can be ground back such that the fused silica capillary fits between the hyper-hemisphere and the hemisphere maintaining the optical scheme, i.e., facilitating positioning of the capillary adjacent to the planar surface(s) at a point of aplanatic focus. The air-space between the hyper-hemisphere and the hemisphere on either side of the capillary is filled with index-matching substance, i.e., gel or liquid. The gel or liquid is index-matched at least to the capillary.
In another embodiment, according to the invention, only a single hyper-hemisphere having a substantially planar surface is used. The capillary is located at an aplanatic point on the single hyper-hemisphere""s substantially planar surface.
Features of the invention include provisions of a spectroscopy system, wherein the precise location of which is relatively insensitive to movement of the hyper-hemisphere/capillary assembly. The system possesses a high tolerance for focusing errors and is implemented as a simple assembly.
The hyper-hemisphere used in the present invention increases the numerical aperture (N.A.) of the system by approximately 50%, thereby increasing the collection efficiency. Further, by effectively forming one optical component with the capillary, it eliminates reflection losses as well as the lensing effect of the outer capillary wall. Additionally, the single hyper-hemisphere approach gives the system a very high tolerance for alignment errors.